The extracellular matrix (ECM) controls a large number of physiological processes including differentiation, apoptosis, and proliferation. Moreover, changes in ECM composition and tissue stiffness are hallmarks of diseases as diverse as fibrosis, cancer, and atherosclerosis. In large part, the ECM regulates cellular function by binding to and activating the integrin family of surface receptors. The necessity of integrin signaling for G1 phase cell cycle progression is now well established, but the approaches that have been used to document ECM/integrin effects typically rely on inhibition of cell adhesion, actin polymerization, or Rho-Rho kinase signaling using cells cultured on rigid plastic or glass surfaces that do not model the deformability of physiological tissue. Since a hallmark of cell-ECM interactions is the ability to assess extracellular stiffness, ECM compliance may be an important determinant of downstream signaling pathways. We have used deformable ECM-coated hydrogels matched to the physiological compliance that cells encounter in vivo to determine how ECM/integrin signaling regulates proliferation physiologically. Our preliminary data show that integrin-dependent cell cycle events have distinct compliance thresholds, and that the tissue compliance characteristic of mammary glands and aortae acts as a cell cycle inhibitor through a selective effect on cyclin D1. The compliance-regulated signaling pathway involves FAK, but is distinguishable from the signaling pathways that we and others have previously implicated in integrin-dependent induction of cyclin D1. We now propose three aims to determine how physiologically relevant changes in ECM compliance regulate the cell cycle. Aim 1 will use ECM-coated hydrogels to determine the signaling mechanisms by which ECM compliance and FAK regulate cyclin D1 gene expression in MEFs and freshly isolated mouse vascular smooth muscle cells (VSMCs). Aim 2 will use the same experimental systems to study a novel and unexpected post- translational effect of ECM compliance on the function of cyclin D1 and activation of cdk4/6. Aim 3 will then test the roles of FAK on tissue compliance and VSMC proliferation in vivo, using newly acquired methodology for fine-wire vascular injury in the mouse. Our combined use of bioengineered substrata, biophysical measurements of tissue elasticity, cell and molecular biology, and in vivo mouse modeling provides us with a powerful interdisciplinary approach for determining how ECM compliance controls integrin signaling to the cell cycle. Since the ECM remodels at sites of vascular injury, the results from these studies may also have important implications for understanding how changes in tissue compliance affect VSMC proliferation in atherosclerosis and restenosis. PUBLIC HEALTH RELEVANCE: One of the main limitations of modern cell biology is that cells are usually cultured on a plastic surface which is completely rigid rather than its native biological substratum which is flexible. The flexibility of an underlying substratum (its "compliance") has profound effects on cellular architecture, differentiation, and proliferation, raising the possibility that some of the signaling, differentiation, and proliferative responses identified in traditional culture may not be relevant in vivo. We have recently adopted a culture system that gives us complete control of substratum compliance and allows us to match the compliance of cultured cells to the compliance of their native tissues. Using this system, we show that physiological tissue compliance is a negative regulator of the cell cycle in vascular smooth muscle cells. We now propose to determine the mechanism underlying this effect. Since the ultimate test of biological relevance must be made in a living organism, this application also exploit a newly acquired mouse model of vascular injury to test the biological relevance of the signaling events that we characterize in compliance-appropriate culture. In addition to the advance in basic cell biology, our proposed work has strong biomedical relevance because smooth muscle cell proliferation and vascular remodeling are critical aspects of both atherosclerosis and restenosis (smooth muscle cell proliferation after balloon angioplasty). Thus, understanding how physiological ECM compliance inhibits vascular smooth muscle cell proliferation, and how this control can be overcome by pathological stiffening of arteries, has the potential to be a significant biomedical advance.